Amphipathic protein-1

ABSTRACT

This invention relates to amphipathic protein-1 polypeptides and genes encoding them. The amphipathic protein-1 polypeptides of the invention protect plants from tissue damage caused by the hypersensitive response, which is often elicited by bacterial infection in higher plants.

BACKGROUND OF THE INVENTION

The hypersensitive response (HR) of higher plants is characterized bythe rapid, localized death of plant cells at the site of pathogeninvasion. HR occurs during incompatible pathogen/host interactions, suchas when a microorganism that normally causes a disease in its host plantinfects a non-host plant. The response is associated with resistanceagainst a variety of pathogens, including nematodes, fungi, viruses, andbacteria. For a review of the hypersensitive response, see Dixon et al.,Annu Rev Phytopathol 32:479 (1994) and Godiard et al., Curr Opin GenetDev 4:662 (1994).

The ability of phytopathogenic bacteria to cause HR in resistant ornon-host plants is controlled by a cluster of highly conserved bacterialgenes named hypersensitive response and pathogenicity (hrp) genes. Mosthrp genes are involved in forming a protein secretion apparatus forharpins, heat-stable and proteinaceous proteins which elicit HR wheninfiltrated into the leaf intercellular spaces of non-host plants. It isknown that, when added to a plant cell culture, harpins induce theexchange of H⁺ and K⁺ across the plasmalemma to generate active oxygenspecies (Baker et al., Plant Physiol 102:1341 [1993]).

SUMMARY OF THE INVENTION

This invention relates to an isolated nucleic acid encoding anamphipathic protein-1 (AP-1) which decreases the extent or duration ofHR in a plant. The term "amphipathic protein-1" refers to any natural orman-made variant of AP-1. For example, the nucleic acid can have thesequence of SEQ ID NO:1 (shown below), have a sequence which hybridizesunder stringent conditions to SEQ ID NO:1, or have a sequence whichencodes SEQ ID NO:2.

A vector and transformed host cell containing such a nucleic acid isincluded within the scope of this invention. A vector is any nucleicacid molecule or virus containing regulatory elements or reporter genesfor the purpose of, but not limited to, expression in prokaryotic oreukaryotic cells or organisms. A transformed host cell is a host cellinto which (or into an ancestor of which) has been introduced, by meansof molecular biological techniques, a nucleic acid encoding an AP-1 ofthis invention. After introduction into the cell, this nucleic acid canexist extrachromosomally or become integrated into the host genome.

The present invention also relates to a substantially pure AP-1, such asSEQ ID NO:2 (shown below) or a protein which differs from SEQ ID NO:2 byat least one conservative amino acid substitution.

A "nucleic acid" encompasses both RNA and DNA, including cDNA, genomicDNA, and synthetic (e.g., chemically synthesized or modified) DNA. Thenucleic acid may be double-stranded or single-stranded. Where singlestranded, the nucleic acid may be a sense strand or an antisense strand.An "isolated nucleic acid" refers to a nucleic acid which may be flankedby non-natural sequences, such as those of a plasmid or virus. Thus, thenucleic acid can include none, some, or all of the 5' non-coding (e.g.,promoter) sequences which are immediately contiguous to the codingsequence. The term, therefore, includes, for example, a recombinant DNAwhich is incorporated into a vector including an autonomouslyreplicating plasmid or virus, or into the genomic DNA of a prokaryote oreukaryote, or which exists as a separate molecule (e.g., a cDNA or agenomic DNA fragment produced by PCR or restriction endonucleasetreatment) independent of other sequences. The term also includes arecombinant DNA or RNA which is part of a hybrid gene encoding anadditional polypeptide sequence. Moreover, the term is meant to includenucleic acid fragments which are not naturally occurring as fragmentsand would not be found in the natural state.

By "hybridizes under stringent conditions" is meant the conditions inwhich a nucleic acid forms a stable, sequence-specific, non-covalentbond with the nucleic acid of SEQ ID NO:1 in solution or on solidsupport under the low salt and high temperature conditions regarded asstringent and set forth in Sambrook et al (Molecular Cloning, ALaboratory Manual, Sambrook, J., Fritsch, E. F., and Maniatis, T., 2nded. [1989] Cold Spring Harbor Laboratory Press). For example, referencenucleic acids such as SEQ ID NO:1 can be immobilized on nitrocellulosefilters. Any nucleic acids specifically and non-covalently binding tothe immobilized reference nucleic acids in the presence of 0.2× SSC(1.75 g/l NaCl, 0.88 g/l Na₃ citrate.2H₂ O; pH 7.0) and 0.1% (w/v)sodium dodecylsulfate at 68° C. are considered to be hybridized understringent conditions.

The term "substantially pure" as used herein in reference to a givenAP-1 polypeptide means that the polypeptide is substantially free fromother compounds, such as those in cellular material, viral material, orculture medium, with which the polypeptide may have been associated(e.g., in the course of production by recombinant DNA techniques orbefore purification from a natural biological source). Polypeptides aresubstantially free from other compounds when they are withinpreparations that are at least 60% by weight (dry weight) the compoundof interest. Preferably, the preparation is at least 75%, morepreferably at least 90%, and most preferably at least 99%, by weight thecompound of interest. Purity can be measured by any appropriate standardmethod, for example, by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

Where a particular polypeptide or nucleic acid molecule is said to havea specific percent identity to a reference polypeptide or nucleic acidmolecule of a defined length, the percent identity is relative to thereference polypeptide or nucleic acid molecule. Thus, a polypeptide thatis 50% identical to a reference polypeptide that is 100 amino acids longcan be a 50 amino acid polypeptide that is completely identical to a 50amino acid long portion of the reference polypeptide. It might also be a100 amino acid long polypeptide which is 50% identical to the referencepolypeptide over its entire length.

For polypeptides, the length of the reference polypeptide sequence willgenerally be at least 16 amino acids, preferably at least 20 aminoacids, more preferably at least 25 amino acids, and most preferably 35amino acids, 50 amino acids, or 100 amino acids. For nucleic acids, thelength of the reference nucleic acid sequence will generally be at least50 nucleotides, preferably at least 60 nucleotides, more preferably atleast 75 nucleotides, and most preferably at least 100 nucleotides(e.g., 150, 200, 250, or 300 nucleotides).

In the case of polypeptide sequences that are less than 100% identicalto a reference sequence, the non-identical positions are preferably, butnot necessarily, conservative substitutions for the reference sequence.Conservative substitutions typically include substitutions within thefollowing groups: glycine and alanine; valine, isoleucine, and leucine;aspartic acid and glutamic acid; asparagine and glutamine; serine andthreonine; lysine and arginine; and phenylalanine and tyrosine. Forexample, a 10 amino acid polypeptide is said to be at least 80%conserved if it differs from a reference polypeptide by no more than twonon-conservative substitutions. An AP-1 polypeptide of this invention ispreferably 70% conserved, more preferably 80% conserved, and mostpreferably 90% conserved as compared to SEQ ID NO:2.

Sequence identity can be measured using sequence analysis software,e.g., the Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705 with the default parameters as specifiedtherein.

The BLAST programs, provided as a service by the National Center forBiotechnology Information, are useful for making sequence comparisons.The programs are described in detail by Karlin et al., Proc Natl AcadSci USA 87:2264 (1990) and 90:5873 (1993), and Altschul et al., NuclAcids Res 25:3389 (1997) and are available on the Internet athttp://www.ncbi.nlm.nih.gov.

The isolation and characterization of an AP-1 gene of the invention willallow the production of variant forms of AP-1 polypeptides which mayhave advantageous activities such as greater HR-blocking activity orgreater protein stability.

Other features or advantages of the present invention will be apparentfrom the following drawings and detailed description of severalembodiments, and also from the claims.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to amphipathic protein-1, a polypeptide whichdecreases the extent or duration of HR in plants, e.g., in response to aharpin secreted from a bacterium.

Contemplated within the scope of this invention are recombinant nucleicacids or viruses which allow production of AP-1 in a transformed cell ortransgenic organism or allow ease of producing specific or non-specificmutations within the AP-1 reading frame. These recombinant nucleic acidsor viruses may further include any one of a variety of sequencesupstream of the AP-1 coding sequences, such as strong constitutivepromoters; within the AP-1 coding sequence, such as introns containingcis-elements that allow high level expression; or downstream of the AP-1coding sequence, such as efficient polyadenylation signals. Theinvention further includes any cells containing or producing suchnucleic acids or viruses, and any AP-1 polypeptides produced from suchcells.

Without further elaboration, it is believed that one skilled in the artcan, based on the above disclosure and the isolation of AP-1polypeptides and genes from the description below, utilize the presentinvention to its fullest extent. The following examples are to beconstrued as merely illustrative of how one skilled in the art canisolate AP-1 genes and polypeptides from biological sources, and are notlimitative of the remainder of the disclosure in any way. Anypublications cited in this disclosure are hereby incorporated byreference.

EXAMPLE 1 Isolation of Amphipathic Protein-1

Three plants were used for the extraction of AP-1. Cotton cultivar(Gossypium hirsutum L.) was obtained from the Jiangsu Academy ofAgricultural Sciences (Nanking, China). Tomato cultivar (Lycopersicumesculentum, Money-maker, CF4) was obtained from Dr. P. J. G. M. De Witat Agricultural University, Wageningen, The Netherlands and is availableas ATCC Nos. 54003 and 54004. Sweet pepper cultivar (Capsicum annuum,ECW ) was obtained from Dr. C-F. Wang in the Asian Vegetable Researchand Developmental Center (AVRDC; Tainan, Taiwan, Republic of China) andis available as AVRDC Accession No. C00165-1.

Healthy leaves from each plant were homogenized in 0.5 M Tris buffer (pH8.0) in the presence of 1% (w/v) polyvinylpyrrolidone and 1.5% (v/v)n-octanol. The homogenates were filtrated by nylon mesh and centrifugedat 10,000 g for 30 minutes at 4° C. The fatty layer at the top of thecentrifuge tubes was collected, resuspended in Tris buffer (0.05 M, pH7.5) containing 1 mM phenylmethylsulfonylfluoride and gently shaken forat least 1 hour. The amphipathic extracts were obtained from the solublephase after centrifugation at 10,000 g for 30 minutes at 4° C.

Amphipathic extracts from the three plants were first separated bySephacryl S-100 gel filtration (Pharmacia Co.) as follows. 6 mg of totalprotein in a volume of 5 ml were loaded in a 1.5×100 cm column (Bio-Rad)and eluted with Tris buffer (50 mM, pH 7.5) at a flow rate of 0.1 ml/minusing the Econo Chromatography system (Bio-Rad). All fractions weretested for their ability to interfere with HR elicitor activity byinfiltrating equal amounts of the eluted proteins and harpin_(Pss) intotobacco plant leaves (procedure described below). Fractions 49-51 fromall plant sources contained significant HR inhibitory activity. Suchfractions were pooled for the next step of purification.

A 1.5×30 cm column (Bio-Rad) containing DEAE Sepharose CL-6B (Pharmacia)was used for further purification. A 0 to 1.0 M NaCl gradient in Trisbuffer (50 mM, pH 7.5) was used to elute proteins at a flow rate of 0.5ml/min, using the Econo Chromatography System. The eluents were desaltedusing a Microconcentrator with 10 kDa molecular weight cut-off membrane(Amicon Co.) and washed three times with phosphate buffer. Fractions26-32 from all plant sources showed significant HR inhibitory activity,which correlated with a single 22 kDa polypeptide as visualized bysubjecting the various fractions to sodium dodecylsulfate-polyacrylamidegel electrophoresis (SDS-PAGE) and subsequent silver staining. The 22kDa protein purified as described above is named amphipathic protein-1or AP-1.

The AP-1 yield from tomato leaves was about 10⁻⁸ gram protein/gram leaf.The yield from sweet pepper or cotton was about 10⁻⁹ gram protein/gramleaf.

The N-terminal amino acids of AP-1 from the three plants weredetermined. AP-1 was isolated, separated by SDS-PAGE, transferred ontoPVDF membranes, and analyzed using a ABI 476A protein sequencer (Appliedbiosystems, Inc.) as instructed by the manufacturer. The first 15 aminoacids of each AP-1 from the three plants were determined as follows:

    ______________________________________                                        Cotton:  Ala Glu Tyr Lys Val Thr Leu Leu Asp Pro Gly Gly                                 Ala Gln Gln (SEQ ID NO:3)                                            Pepper: Ala Thr Tyr Lys Val Lys Leu Val Thr Pro Asp Gly                        Pro Val Glu (SEQ ID NO:4)                                                    Tomato: Ala Thr Tyr Lys Val Lys Leu Ile Thr Pro Glu Gly                        Pro Phe Phe (SEQ ID NO:5)                                                  ______________________________________                                    

EXAMPLE 2 AP-1 Delays Harpin-Induced Hypersensitive Response

To test the effect of AP-1 on Harpin induced HR, Harpin_(Pss) (harpinfrom Pseudomonas syringae pv. syringae) protein was first prepared.Harpin_(Pss) protein was extracted by the methods described in He etal., Cell 73:1255 (1993). E. coli DH5α (pSYH10) containing theharpin_(Pss) gene was grown in Lauria Broth containing ampicillin (50μg/ml) at 37° C. in the dark and shaken overnight in the presence ofisopropyl-β-D-thiogalactoside (IPTG). To obtain harpin_(Pss), thebacteria were washed, sonicated for 30 seconds, and boiled for 10minutes. After boiling, the extracts were centrifuged at 10,000 g for 10minutes. Supernatants were desalted by Microconcentrators (Amicon) andwere stored at 4° C.

The HR assay was performed as described in Huang et al., J Bacteriol170:4748 (1988). Fully expanded tobacco leaves (Nicotiana tabacum L. cv.Xanthi, available as ATCC Nos. 54037 and 54039) were wounded with a 25gauge needle to form tiny holes on the lower surfaces of the leaves.Harpin_(Pss) or bacterial cells was infiltrated by pressing a 1 ml bluntsyringe through the hole. The infiltrated plant was incubated in a 28°C., 12 hour light/12 hour dark incubator. The HR was recorded byphotography.

Various amounts of purified AP-1 were mixed with 10 μg of harpin_(Pss),and the resulting mixture was infiltrated into the intercellular spacesof tobacco leaves. The area of necrosis 20 hours after infiltration inthe presence of as little as 50 ng of tomato AP-1 was reduced incomparison with the absence of AP-1.

AP-1 also caused a significant postponement of HR-like necrosis inducedby harpin_(Pss) -harboring bacteria. Bacteria was infiltrated in theabsence or presence of 500 ng of tomato AP-1 into the intercellularspaces of the tobacco leaf at the equivalent of 5×10⁶ CFU/ml. At sixdays post-inoculation, the bacteria caused significant necrosis in theabsence of AP-1. In the presence of AP-1, the infiltrated plant tissuewas relatively healthy.

The HR reduction rate was approximately proportional to the dosage ofAP-1 in the range from 50 ng to 500 ng and approached 80% reduction ofHR at the highest dose at 20 hours post-inoculation. 250 ng of AP-1,which corresponds to a harpin_(Pss) to AP-1 molar ration of 40 to 1,established about a 50% reduction of the HR-necrosis area.

A time-course of AP-1 infiltration was performed with respect to P.syringae inoculation. HR was not delayed when AP-1 was infiltrated 30 or60 minutes prior to inoculation. In contrast, AP-1 delayed HRsignificantly when AP-1 and bacteria were infiltrated simultaneously.

EXAMPLE 3 AP-1 Suppresses Bacterial Growth

P. syringae pv. syringae was infiltrated, along with tomato AP-1, intothe intercellular spaces of tobacco leaves. The bacteria population wasmonitored as described in Li et al., J Bacteriol 174:1742 (1992). 1×10⁷cfu/ml of log-phase growth bacteria was used for each inoculation. Leafdisks (0.5 cm diameter) punched around the infiltration holes werehomogenized in 100 μl phosphate buffer (0.01 M, pH 6.5) and diluted upto 10,000 fold. 100 μl of bacteria were plated on 9 cm LB agar plates,and the colonies counted after overnight growth. Each dilution wasplated in triplicate.

Within 48 hours post-inoculation, the population of the bacteria and thebacteria treated with 1 μg of bovine serum albumin (BSA) steadilyincreased from 10⁵ CFU/ml to 10⁷ CFU/ml per leaf disk. However, in thepresence of 1 μg of AP-1, the bacterial population was reduced from aninitial 5×10⁵ CFU/ml to 5×10⁴ CFU/ml 16 hours post-inoculation.

EXAMPLE 4 Cloning of an AP-1 CDNA

Total RNA was isolated from pepper leaves (Capsicum annuum) as describedin Nelson, "Preparation of DNA and RNA from leaves: expanded blades andseparated bundle sheath and mesophyll cells" In The Maize Handbook,Freeling et al. editors, pp. 541-545, Springer-Verlag, NY, 1994. Twograms of pepper leaves were homogenized, using a Pro 200 homogenizer, in10 ml of extraction buffer (0.1 M TRIS-HCl, pH 8.5, 0.1 M NaCl, 20 mMEDTA, and 1% lauroyl sarcosine). RNA was separated from DNA byprecipitation with 2 M LiCl. First-strand cDNA synthesized using anoligo (dT) primer and Superscript Reverse Transcriptase (GibcoBRL/Life)according to the manufacturer's instructions.

For PCR, 2 μl of RT mixture was combined with 0.6 μM of modifieddegenerate primer (5'-GCiACiTAYAARGTiAAR-3'; [SEQ ID NO:6]), 0.2 μM of5'-(dT)N anchor primer, 1 mM dNTP, and 2.5 units of Taq DNA polymerase(GibcoBRL/Life). The sequence of PCR primers used to clone AP-1 werederived from the N-terminal amino acid sequence as described in Example1 above. The amplification cycle parameters were as follows: 5 minutesat 94° C.; two cycles of 1 minute at 94° C., 2 minutes at 45° C., 1.5minutes at 72° C.; 38 cycles of 45 seconds at 94° C., 2 minutes at 63°C., 1 minute at 72° C.; and 10 minutes at 72° C. PCR products werepurified using a spin-column with a silica-gel binding membrane(Qiagen).

The second, nested PCR was amplified by mixing together 1 μl of theabove purified PCR products, 0.4 μM of modified degenerate primer(5'-ACiCCiGAYGGiCC-3'; SEQ ID NO:7), 0.4 μM of 3' oligo 5' (dT)₁₈ Nanchor primer and otherwise treated as indicated immediately above.

RT-PCR products were run on an agarose gel and eluted using a Qiaquickgel extraction kit (Qiagen). The purified cDNA fragments were treatedwith polynucleotide kinase and ligated into the pT7Blue blunt-end vector(Novagen) according to manufacturer's instructions.

The sequence of cDNA inserts was determined by the dideoxy chaintermination method using a Sequenase kit (PE-ABI) and an ABI nucleicacid sequencer.

To amplify the 5' Cap region of the gene, first-strand cDNA wassynthesized by performing RT-PCR (GibcoBRL/Life) in the presence of 0.5μM Capswitch primer (Clontech) and 800 ng pepper mRNA. mRNA was isolatedfrom total pepper RNA using oligo (dT) affinity magnetic particles(Straight-A mRNA Isolation System, Novagen). PCR amplification of the 5'cap region was performed in a 30 μl volume containing 2 μl single-strandcDNA, the 5' Capswitch primer, and 3'-specific primer(5'-CATCTTGGTCAAAGTTTGAATC-3'; SEQ ID NO:8) corresponding to the 3'noncoding region of the AP-1 clone. Amplification was performed usingthe following parameters: 35 cycles of 94° C. for 45 seconds, 53° C. for1 minute, and 72° C. for 1 minute; and 5 minutes at 72° C. Following PCRamplification, the Klenow cDNA fragments were purified, treated withpolynucleotide kinase, and ligated into pT7Blue Blunt vector (Novagen).

A 785 base pair full-length cDNA clone (pap-1), containing N-terminaldeduced amino acid sequences of the putative mature peptide, wasisolated. This clone encodes a polypeptide with the same N-terminalamino acids as described in Example 1 above. The pap-1 cDNA clonecontains an open reading frame (ORF) of 432 bp, encoding a protein of144 amino acids, which includes a 47 amino acid putative secretionpeptide. The coding sequence of the open reading frame is shown below.

    __________________________________________________________________________    ATGGCTTCATACAAAGTGAAACTTATCACACCTGACGGACCAATAGAATTTGATTGCCCA                    GATAATGTGTACATTCTTGATCAAGCTGAGGAAGCAGGACATGATCTTCCTTATTCGTGC                  AGGGCAGGTTCTTGCTCATCTTGTGCTGGTAAAATTGCTGGTGGAGCTGTTGATCAAACT                  GATGGCAACTTTCTTGATGATGACCAATTAGAGGAGGGATGGGTGCTAACTTGTGTTGCT                  TATCCACAGTCTGATGTTACTATTGAGACTCACAAAGAGGCAGAACTCGTGGGC                        (SEQ ID NO:1)                                                               __________________________________________________________________________

The deduced amino acid sequence contains a 2Fe--2S domain, amyristoylation site, and two phosphorylation sites. The AP-1 amino acidsequence is shown below.

    ______________________________________                                        Met Ala Ser Val Ser Ala Thr Met Ile Ser Thr Ser Phe Met Pro                     Arg Lys Pro Ala Val Thr Ser Leu Lys Pro Ile Pro Asn Val Gly                   Glu Ala Leu Phe Gly Leu Lys Ser Ala Asn Gly Gly Lys Val Thr                   Cys Met Ala Ser Tyr Lys Val Lys Leu Ile Thr Pro Asp Gly Pro                   Ile Glu Phe Asp Cys Pro Asp Asn Val Tyr Ile Leu Asp Gln Ala                   Glu Glu Ala Gly His Asp Leu Pro Tyr Ser Cys Arg Ala Gly Ser                   Cys Ser Ser Cys Ala Gly Lys Ile Ala Gly Gly Ala Val Asp Gln                   Thr Asp Gly Asn Phe Leu Asp Asp Asp Gln Leu Glu Glu Gly Trp                   Val Leu Thr Cys Val Ala Tyr Pro Gln Ser Asp Val Thr Ile Glu                   Thr His Lys Glu Ala Glu Leu Val Gly                                           (SEQ ID NO:2)                                                               ______________________________________                                    

To test for the distribution of ap-1 in various plants, genomic DNA wasisolated from pepper, tomato, tobacco, cotton, Arabidopsis thaliana andPetunia sp. according to Nelson, Id. For PCR detection of ap-1sequences, a 5' primer in the ap-1 coding sequence(5'-AATAGAATTTGATTGCCCAGA-3'; SEQ ID NO:9) and 3' primer(5'-CATCTTGGTCAAAGTTTGAATC-3'; SEQ ID NO:8) in the 3' untranslatedregion were used. The 5' primer and the 3' primer was expected toproduce a 373 bp PCR product. The PCR was performed in MgCl₂ buffercontaining 1 mM dNTP, 2.5 units of Taq DNA polymerase, 200 ng genomicDNA and 5% dimethylsulfoxide. The amplification parameters were asfollows: 5 minutes at 94° C.; 5 cycles of 1 minute at 94° C., 1 minuteat 51° C., and 1 minute at 72° C.; and 30 cycles of 45 seconds at 94°C., 1 minute at 51° C., and 1 minute at 72° C.; and 5 minutes at 72° C.

A 373 bp fragment could be amplified from pepper, tomato, tobacco,Petunia, and cotton genomic DNA but not from Arabidopsis genomic DNA.

EXAMPLE 5 In Vitro Expression of Cloned AP-1

In order to transiently express pepper AP-1 in vivo, the papl codingregion was subcloned into a bamboo mosaic potexvirus satellite (satBaMV)vector, which is described in Lin et al., Proc Natl Acad Sci USA 93:3138(1996), hereby designated BSAP1. BASP1 was used to in vitro transcribean RNA containing the 432 bp ap-1 open reading frame and about 400 bp ofviral sequence. The BaMV-L and satBaMV vector transcripts wereinoculated, along with the ap-1 transcript, into the tobacco Nicotianabenthamiana. Northern blot hybridization was used to detect thereplication of viral RNA in tobacco. The total RNA extracted 7 daysafter inoculation was separated on 1% agarose gels, transferred to nylonmembranes, and hybridized with a probe specific to the 3' end of satBaMV(+)RNA as described in Lin et al., (1996) Id. The Northern indicatedthat the BSAP1 RNA was expressed in the tobacco.

For in vitro translation, the satBaMV vector transcripts were translatedin rabbit reticulocyte lysate (Promega) in the presence of ³⁵ S-labeledmethionine and analyzed by SDS-PAGE. The in vitro translation produced a27 kDa protein. The size of the translated protein was about two foldslarger than the predicted molecular weight of the api encodedpolypeptide (14 kDa), including the putative secretion peptide region.

For expression of AP-1 in E. coli, the mature polypeptide of the pap1clone was cloned into the Bam HI and Hind III sites of the pQE-30 vector(Qiagen) and transformed into the bacterial host strain M15. TheN-terminal 6× His-tagged proteins were induced with IPTG and purified byNi-NTA resin Spin Kits (Qiagen). For immunoblot detection of the 6×His-tagged AP-1 polypeptides, the E. coli lysate was run on SDS-PAGEgels, transferred onto nitrocellulose membranes, and detected with.^(RGS) His antibodies (Qiagen).

In the above system, the translated protein, which does not include thesecretion peptide, was also observed to be double the size (22 kDa) ofthe predicted molecular weight of AP-1. This size, however, isconsistent with the estimated molecular weight of the plant AP-1polypeptide as described in Example 1 above.

EXAMPLE 6 Cloned AP-1 Delays Harpin_(Pss) -mediated HR

To determine the biological activity of cloned AP-1, a mixture of thepurified harpin_(Pss) and E. coli expressed pepper AP-1, as obtained inExample 5 above, was infiltrated into tobacco leaves as described inExample 2 above. A light yellowing response was induced by harpin_(Pss)within one day post-inoculation and was followed by the formation ofharpin_(Pss) -induced necrosis two days post-inoculation. The necrosisarea was reduced more than 50% two days post-inoculation when 200 ngAP-1 was co-inoculated with the harpin.

EXAMPLE 7 Cloned AP-1 Suppresses HR induced by Erwinia carotovora subsp.carotovora

Fifty microliters containing different concentrations of the pathogenicbacteria Erwinia carotovora subsp. carotovora in the presence or absenceof 0.15 μM AP-1 were inoculated into tobacco leaves and assayed for HRinhibition as described in Example 2 above. E. carotovora subsp.carotovora can be obtained as ATCC No. 12312.

AP-1 protected the plant tissue from E. carotovora-induced HR necrosisat one day post-inoculation for bacterial concentrations up to 10⁷CFU/ml.

EXAMPLE 8 Agrobacterium-Mediated Overexpression of AP-1 Suppresses HRinduced by Xanthomonas campestris pv. vesicatoria

Agrobacterium-mediated transient overexpression of sweep pepper AP-1 wasestablished in sweet pepper plants as described in Van den Ackerveken etal., Cell 87:1307 (1996). Agrobacterium bacteria was infiltrated intofully expanded leaves of the sweet pepper overexpressing AP-1. Plantswere kept for two days at 20° C., 16 hour light/8 hour dark, 100%humidity and then moved to 28° C., 16 hour light/8 hour dark, 80%humidity. Fifty microliters of Xanthomonas campestris pv. vesicatoriasuspension at 1×10⁴ CFU/ml was inoculated eight days after theAgrobacterium inoculation in the same region and incubated for 24 hours.X. campestris pv. vesicatoria can be obtained as ATCC Nos. 11551 and11633.

The Agrobacterium-mediated expression of AP-1 prevented HR necrosisinduced by the pathogenic X. campestris.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thisinvention.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 9                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 294 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                    (ii) MOLECULE TYPE: cDNA                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - -  ATGGCTTCAT ACAAAGTGAA ACTTATCACA CCTGACGGAC CAATAGAATT - #TGATTGCC    CA    60                                                                        - -  GATAATGTGT ACATTCTTGA TCAAGCTGAG GAAGCAGGAC ATGATCTTCC - #TTATTCGTG    C   120                                                                         - -  AGGGCAGGTT CTTGCTCATC TTGTGCTGGT AAAATTGCTG GTGGAGCTGT - #TGATCAAAC    T   180                                                                         - -  GATGGCAACT TTCTTGATGA TGACCAATTA GAGGAGGGAT GGGTGCTAAC - #TTGTGTTGC    T   240                                                                         - -  TATCCACAGT CTGATGTTAC TATTGAGACT CACAAAGAGG CAGAACTCGT - #GGGC             294                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 144 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - -  Met Ala Ser Val Ser Ala Thr Met Ile Ser - #Thr Ser Phe Met Pro Arg         1               - #5                  - #10                  - #15          - -  Lys Pro Ala Val Thr Ser Leu Lys Pro Ile - #Pro Asn Val Gly Glu Ala                    20    - #              25    - #              30                 - -  Leu Phe Gly Leu Lys Ser Ala Asn Gly Gly - #Lys Val Thr Cys Met Ala                35        - #          40        - #          45                     - -  Ser Tyr Lys Val Lys Leu Ile Thr Pro Asp - #Gly Pro Ile Glu Phe Asp            50            - #      55            - #      60                         - -  Cys Pro Asp Asn Val Tyr Ile Leu Asp Gln - #Ala Glu Glu Ala Gly His        65                - #  70                - #  75                - #        80                                                                               - -  Asp Leu Pro Tyr Ser Cys Arg Ala Gly Ser - #Cys Ser Ser Cys Ala        Gly                                                                                               - #85                  - #90                  - #95         - -  Lys Ile Ala Gly Gly Ala Val Asp Gln Thr - #Asp Gly Asn Phe Leu Asp                   100     - #            105     - #            110                 - -  Asp Asp Gln Leu Glu Glu Gly Trp Val Leu - #Thr Cys Val Ala Tyr Pro               115         - #        120         - #        125                     - -  Gln Ser Asp Val Thr Ile Glu Thr His Lys - #Glu Ala Glu Leu Val Gly           130             - #    135             - #    140                         - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - -  Ala Glu Tyr Lys Val Thr Leu Leu Asp Pro - #Gly Gly Ala Gln Gln             1               - #5                  - #10                  - #15          - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - -  Ala Thr Tyr Lys Val Lys Leu Val Thr Pro - #Asp Gly Pro Val Glu             1               - #5                  - #10                  - #15          - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - -  Ala Thr Tyr Lys Val Lys Leu Ile Thr Pro - #Glu Gly Pro Phe Phe             1               - #5                  - #10                  - #15          - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: primer, degenerate                                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: synthetic - #oligonucleotide                                    (B) LOCATION: 3, 6, - #and 15                                                 (D) OTHER INFORMATION: - #where N at positions 3, 6, and 15 is                     inosine                                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - -  GCNACNTAYA ARGTNAAR            - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: primer, degenerate                                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: synthetic - #oligonucleotide                                    (B) LOCATION: 3, 6, - #and 12                                                 (D) OTHER INFORMATION: - #where N at positions 3, 6, and 12 is                     inosine                                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - -  ACNCCNGAYG GNCC             - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: primer                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - -  CATCTTGGTC AAAGTTTGAA TC          - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: primer                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - -  AATAGAATTT GATTGCCCAG A          - #                  - #                      - #21                                                                 __________________________________________________________________________

What is claimed is:
 1. An isolated nucleic acid comprising SEQ ID NO:1.2. A vector containing the nucleic acid of claim
 1. 3. A transformedhost cell containing the nucleic acid of claim
 1. 4. An isolated nucleicacid comprising a sequence which encodes SEQ ID NO:2.
 5. A vectorcontaining the nucleic acid of claim
 4. 6. A transformed host cellcontaining the nucleic acid of claim
 4. 7. An isolated nucleic acidencoding an amphipathic protein-1, the nucleic acid hybridizing to thecompliment of a reference nucleic acid consisting of SEQ ID NO:1,wherein the hybridization is performed in the presence of 0.2× SSC and0.1% (w/v) sodium dodecylsulfate, and at 68° C.
 8. The nucleic acid ofclaim 7, wherein the amphipathic protein-1 decreases the growth of abacterium in a plant.
 9. The nucleic acid of claim 7, wherein theamphipathic protein-1 is derived from a cotton, pepper, tobacco, ortomato plant.